抗冷冻蛋白Ⅲ对小鼠胚胎冷冻保护的作用

Exploration of the protective role of antifreeze protein Ⅲ on mouse embryo in vitrification

目的将抗冷冻蛋白III(AFPIII)应用于小鼠2-细胞期胚胎玻璃化冷冻保存,观察其对细胞微丝骨架结构和冷冻复苏后发育潜能是否有保护作用。方法收集小鼠2-细胞期胚胎1 200枚,随机分为新鲜对照组、玻璃化冷冻组和添加AFPIII玻璃化冷冻组(AFPIII终浓度为500 ng/mL),每组400枚,其中200枚胚胎进行复苏后囊胚培养,200枚进行细胞微丝的免疫荧光染色,比较各冷冻组胚胎复苏存活率和其后的囊胚形成率以及各组微丝完整胚胎比率。结果添加AFPIII玻璃化冷冻组胚胎复苏率高于玻璃化冷冻组,差异有统计学意义(P<0.05)。新鲜对照组和添加AFPIII玻璃化冷冻组囊胚形成率、微丝完整胚胎比率均高于玻璃化冷冻组,差异有统计学意义(P<0.05);新鲜对照组和添加AFPIII玻璃化冷冻组之间差异无统计学意义(P>0.05)。结论 AFPIII对小鼠2-细胞期胚胎玻璃化冷冻有保护作用。

Objective To observe whether antifreeze protein Ⅲ（AFPⅢ） is able to protect cytoskeleton microfilament and the potential development after recovery while being used in the vitrification of two-cell stage embryos. Methods A total of 1 200 two-cell stage embryos were collected and randomly divided into 3 groups： fresh control group,vitrification group,and vitrification group with additional AFPⅢ（500 ng/mL） group,with 400 embryos in each group. Half of the embryos in each group were used for blastocyst culture after recovery,and the left were used for immunofluorescent staining of the microfilament,to compare the recovery rate,the following blastocyst formation rate,and the rate of formal cytoskeleton microfilament. Results The recovery rate in the vitrification group with additional AFPⅢ was higher than that of the vitrification group（P ＆lt; 0. 05）; the ratio of blastocyst formation rate and intact actin filaments were higher in the fresh control group and vitrification group with additional AFPⅢ,compared to that of the vitrification group（both P ＆lt; 0. 05）; there was no statistical difference between the fresh control group and vitrification group with additional AFPⅢ（P ＆gt; 0. 05）. Conclusion AFPⅢ plays a protective role on mouse two-stage embryos in vitrification.